CN105218659B - Human BMP-7 mature peptide and expression thereof - Google Patents
Human BMP-7 mature peptide and expression thereof Download PDFInfo
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- CN105218659B CN105218659B CN201510731405.7A CN201510731405A CN105218659B CN 105218659 B CN105218659 B CN 105218659B CN 201510731405 A CN201510731405 A CN 201510731405A CN 105218659 B CN105218659 B CN 105218659B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Does the present invention provide human BMP-7, and it is mature peptide, and its aminoacid sequence is such as SEQ? ID? shown in NO:2 or at SEQ? ID? sequence shown in NO:2 is added, lacks and/or replace one or several amino-acid residue and obtain. Present invention also offers the pharmaceutical composition comprising this human BMP-7, break up calcification for short bone.
Description
Technical field
The invention belongs to protein or technical field of polypeptide, specifically, the present invention relates to human BMP-7 (especially restructuring produces, referred to as rhBMP2) and medicine and the differentiation calcification application of short bone etc.
Background technology
Bone morphogenetic protein (BMP), also known as human bone morphorgenetic protein, except Human Bone Morphogenetic Proteins-4 1 (BMP1), all the other all belong to transforming growth factor-beta superfamily, that wherein study more is human BMP-7 (BMP2), such as, No. 98113048th, Chinese patent application discloses the rhBMP-2 being used for the treatment of hematopoietic disorder and damage, and BMP2 wherein is wild-type protein; Chinese patent application No. 200610032800 expression method disclosing a kind of recombinant human bone morphorgenetic protein-2, BMP2 wherein is also wild-type protein; No. 201410310953rd, Chinese patent application discloses a kind of recombinant human bone morphogenesis protein-2 truncate and optimization expression method thereof, and the albumen wherein obtained has 115 amino-acid residues; No. 200910045832nd, Chinese patent application also discloses a kind of recombinant human bone morphorgenetic protein-2 truncate and optimization expression method thereof, and the albumen wherein obtained has 114 amino-acid residues, and its output is the highest at about 10mg/L.
But, except the present inventor, people continues to study the BMP2 that more truncates never again. The present inventor is not by the impact of prior art trend, many decades is devoted to the development research of BMP2 (especially rhBMP2), under the limit truncated, develop very unexpectedly again a kind of new human BMP-7, the ability of its short bone differentiation calcification improves, thus there is better bone forming activity/curative effect, have more actual medicinal promotion prospect. In addition, the present inventor have also developed the encoding gene of a kind of new human BMP-7, and it has jumped out existing codon optimized framework, obtains brand-new encoding gene, can significantly improving expression amount in intestinal bacteria, the industrialization being applicable to very much genetically engineered is produced.
Summary of the invention
The technical problem to be solved of the present invention is to provide new human BMP-7 and medicine and the differentiation calcification application of short bone etc. In addition, present invention also offers the gene engineering preparation method of human BMP-7 and polynucleotide thereof etc.
Specifically, in first, the present invention provides human BMP-7, its aminoacid sequence
(1) as shown in SEQIDNO:2; Or
(2) add in sequence shown in SEQIDNO:2, lack and/or replace one or several amino-acid residue and obtain, and the ability of its short osteocyte differentiation calcification improves.
The aminoacid sequence of the human BMP-7 of the first aspect of the invention can be the aminoacid sequence adding in sequence shown in SEQIDNO:2, lacking and/or replace one or several (preferably one to five, it is more preferable to one to three) amino-acid residue and obtain. those skilled in the art know, by changing the coding gene sequence of known peptide and imported expression vector, can prepare replacement, interpolation or lack the polypeptide of amino-acid residue, these methods are extensively recorded in the documents well known in the art such as " molecular cloning experiment guide " (Beijing: scientific publication society, 2002). in the amino-acid residue replaced, it is preferable that be substituted by other amino acid similar to original acid residue side chain character, thus more kept original function active. the similar amino acid of side chain character has hydrophobic amino acid (A respectively, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), amino acid (the G of aliphatic lateral chain, A, V, L, I, P), amino acid (the S of hydroxyl side chain, T, Y), amino acid (the C of sulfur atom-containing side chain, M), containing the amino acid (D of carboxylic acid and amide side chains, N, E, Q), containing the amino acid (R of basic group side chain, K, H), containing the amino acid (H of aromatic series side chain, F, Y, W). in the specific embodiment of the present invention, the aminoacid sequence of human BMP-7 increases methionine(Met) (M) residue as shown in SEQIDNO:2 or at its N end, and it, through national authority Department certification, meets regulation.
The human BMP-7 of preferred the first aspect of the invention improves relative to the short bone differentiation calcification of other the BMP2 truncated. Wherein, other the BMP2 truncated comprises the BMP2 of No. 201410310953rd, Chinese patent application and No. 200910045832nd, Chinese patent application. The short bone differentiation calcification effect of the human BMP-7 of preferred the first aspect of the invention has lifting compared with the BMP2 of No. 201410310953rd, Chinese patent application He No. 200910045832nd, Chinese patent application.
In the 2nd, the present invention provides polynucleotide, the human BMP-7 of first aspect of its code book invention. The polynucleotide of the present invention, it is possible to be DNA form, it is also possible to be rna form, it is preferable that DNA form. DNA form comprises the cDNA of natural cDNA and synthetic, and DNA can be coding strand or template strand. By routine techniques, such as the method for PCR method, recombination method or synthetic, those skilled in the art can obtain nucleic acid molecule or its fragment of the polypeptide of first aspect of code book invention. These sequences once obtain, so that it may to be cloned into carrier, then transform or be transfected into corresponding cell, then by cultivate host cell breed. The nucleotide sequence of the polynucleotide of preferred the present invention is as shown in SEQIDNO:1. This preferred sequence by the conventional restriction expressing optimizing codon, optimizes the expression in intestinal bacteria, and expression effect has and more significantly promotes.
In the 3rd, the present invention provides a kind of carrier, and it contains the polynucleotide of the present invention the 2nd described in aspect. carrier herein comprises expression vector, such as, and bacterium plasmid, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector. the carrier being suitable in the present invention includes but not limited to: the carrier (prokaryotic expression carrier) expressed in bacterium, the carrier expressed in yeast is (such as pichia vector, debaryomyces hansenii carrier etc.), at the baculovirus vector of expressed in insect cells, carrier (the vaccinia virus vector expressed in mammalian cell, retroviral vector, adenovirus carrier, adeno-associated virus carrier etc.), the plant viral vector expressed in plant and the various carriers expressed in mammal galactophore. in a word, as long as duplication can be stablized in host cell, any plasmid and carrier all can use. preferred expression carrier comprises selectable marker gene, such as the ampicillin resistance gene of bacterium, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene, chloramphenicol resistance gene, the neomycin resistance gene of yeast, Zeocin resistant gene, the defect selection marker of yeast, such as His, Leu, Trp etc., the neomycin resistance gene of eukaryotic cell, Zeocin resistant gene, dihydrofolate reductase gene and fluorescent protein marker gene etc. the carrier of the present invention is preferably prokaryotic vector, it is more preferable to be coli expression carrier.
In the 4th, the present invention provides cell, and it contains the polynucleotide of the present invention the 2nd described in aspect. This cell can obtain with the vector described in third aspect of the present invention or transfection. Cell can be prokaryotic cell prokaryocyte, it is also possible to be eukaryotic cell, e.g., and bacterial cell, yeast cell, vegetable cell, insect cell, mammalian cell etc. Cell, after conversion or transfection contain the gene order of encoding fusion protein of the present invention, namely forms through engineering approaches cell or cell strain, can be used for producing required fusion rotein. Suitable conversion or transfection method include but not limited to: for bacterial cell, such as Calcium Chloride Method, electroporation; For yeast cell, such as electroporation and protoplastis fusion method; For mammalian cell etc., such as plastid parcel, coprecipitation of calcium phosphate, electro fusion method and microinjection. The cell of preferred the present invention is Bacillus coli cells.
In the 5th, the present invention provides the method for the human BMP-7 of preparation the first aspect of the invention, cultivate the cell of the present invention the 4th described in aspect when it is included in applicable protein expression, from culture, then isolate the polypeptide of the first aspect of the invention. The method of separation includes but not limited to: split bacterium (ultrasonic wave splits bacterium, infiltration pressure break bacterium), centrifugal, saltout, molecular sieve chromatography (is also called molecular dimension exclusion chromatography), ion-exchange chromatography, adsorption chromatography (affinity chromatography, metal chelate affinity chromatography), reverse chromatograms, high performance liquid chromatography, capillary electrophoresis, isoelectrofocusing and sex change/renaturation process etc.
In the 6th, the present invention provides pharmaceutical composition, and it comprises the human BMP-7 of the first aspect of the invention and pharmaceutically acceptable carrier. " pharmaceutical composition " used herein and " medicine ", it is possible to exchanging and use, if do not indicated especially, what refer to is all pharmaceutical composition and the medicine of people. Pharmaceutically acceptable carrier used herein refers to nontoxic weighting agent, stablizer, thinner, adjuvant or other pharmaceutical adjuncts. The technician of this area can according to therapeutic purpose, route of administration (as injection or oral) need to make pharmaceutical composition various formulation, preferred said composition is unit dosage form, such as freeze-dried, tablet, capsule, pulvis, emulsion agent, injection or sprays, more preferably this pharmaceutical composition is injection formulations, such as freeze-dried or injection liquid. Preferred pharmaceutically acceptable carrier can be physiological saline. This pharmaceutical composition may be used for the differentiation calcification of short bone.
In the 7th, the present invention provides the human BMP-7 of the first aspect of the invention in the application prepared in the medicine urging osteocyte differentiation calcification. This application can be that the human BMP-7 of the first aspect of the invention is separately in the application preparing in medicine, it is also possible to is that associating other short osteocytes differentiation calcification activity composition is in the application preparing in medicine. In addition wherein, the activity/curative effect of short osteocyte differentiation calcification can improve relative to the short osteocyte differentiation calcification/curative effect of No. 201410310953rd, Chinese patent application and the BMP2 of No. 200910045832nd, Chinese patent application. In the specific embodiment of the present invention, osteocyte is marrow stromal cell.
The useful effect that the present invention obtains is: the impact truncating the limit do not studied by the BMP2 of prior art, develops the rhBMP2 made new advances, and its short osteocyte differentiation calcification activity/curative effect improves, and is more suitable for actual popularization medicinal; It has jumped out existing codon optimized framework, obtains brand-new optimization encoding gene, it is possible to significantly improve expression amount in intestinal bacteria, and the industrialization being applicable to very much genetically engineered is produced.
For the ease of understanding, the present invention refer to open source literature, and these documents are to more clearly describe the present invention, and its entire contents is all included in and carried out reference herein.
Hereinafter by specific embodiment, the present invention will be described in detail. It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention. According to the discussion of this specification sheets, many changes, the change of the present invention have been all obviously concerning one of ordinary skill in the art.
Embodiment
Following examples will specifically describe the present invention, wherein if any not detailed part, can carry out see the method described in the laboratory manual such as " molecular cloning experiment guide ", " cell experiment guide ", or experimentally in reagent, the specification sheets that provides of manufacturer of instrument or the handbook specifically used carry out.
The expression of the rhBMP2 of embodiment 1 the present invention
The present inventor has found the rhBMP2 of aminoacid sequence as shown in SEQIDNO:2, and well-designed its is expressed the encoding gene (Nucleotide is as shown in SEQIDNO:1) optimized and prepared at expression in escherichia coli. In brief, entrust Shanghai Jie Rui Bioisystech Co., Ltd this gene of synthetic, then connect between the restriction enzyme site of EcoRI and HindIII of pEVT carrier (NewEnglandBiolabs company), it is transformed into e. coli bl21 (DE3), in containing screening on the LB flat board of penbritin 50mg/L, the expression vector comprised in the bacterium that screening is positive is named as pEVT-hBMP2, extracting plasmid, and order-checking qualification is correct.
Getting positive bacteria is inoculated in 5mLLB substratum, with 30 DEG C, 200r/min cultivate 12h, then to be inoculated in 400mLLB substratum by 2% (V/V) inoculum size, with 30 DEG C, 200r/min cultivate 8h. Then ferment, the 400mL ferment-seeded bacterium of above-mentioned cultivation is inoculated that (often liter contains: peptone 5g, glycerine 5mL into 8L fermention medium, disodium hydrogen phosphate 6g, potassium primary phosphate 1.5g, ammonium sulfate 1.5g, ammonium chloride 1g, bitter salt 0.25g, calcium chloride 0.02g, ferrous sulfate 0.04g, glycine 0.5g, pH7.0) in, utilize NBSBiofloIV20L fermentor tank to ferment, fermentation whole process 20h altogether, period pH controls all the time 7.0��7.2; Dissolved oxygen controls 40% (air flow quantity is set as 12L/min, and stirring velocity is 200r/mim��1000r/min); As bacterial density OD600When reaching 20, the IPTG adding 0.35mM carries out abduction delivering; Before not adding IPTG, when stirring with minimum stirring velocity, dissolved oxygen is still continuing to reach 60% or more in 3min, (often liter contains: glycerine 120mL then to add feed-batch culture base, peptone 50g, yeast extract 50g, bitter salt 2g, pH7.0) until dissolved oxygen returns to 40%; After adding IPTG, when stirring with minimum stirring velocity, dissolved oxygen still continuing to reach 50% or more in 3min, then adds above-mentioned feed-batch culture base until dissolved oxygen returns to 40%. Detect with 15%SDS-PAGE after sampling molten bacterium, have great expression at 14kDa.
After centrifugal collection thalline, the urea 150mL sex change that every 10g weight in wet base thalline adds 8 mol/L is dissolved, under ice bath, stir cracking 8 hours, get supernatant liquor, adopt reversed phase chromatography purifying (separating medium: SOURCE30RPC, at pH8.5, when 6M urea, with 0��40% Virahol gradient elution purifying), (separating medium: SOURCE30Q is purified again with anion column ion chromatography, at pH8.5, when 8M urea, with 0��1mol/L sodium-chlor gradient elution purifying), the respective peaks of purifying gained is collected with SDS-PAGE detection, then add renaturation solution (to contain: 2 mol/L urea, 0.1% (V/V) TritonX100, 1mM reduced glutathione, 1 grams per liter Macrogol 4000, with 5% (V/V) glycerine, pH8.5) renaturation 24h in, (separating medium: SOURCE30Q is purified again with anion-exchange chromatography, at pH8.5, when 1.5M urea, with 0��1mol/L sodium-chlor gradient elution, (separating medium: Sephacryls100 is purified again with molecular exclusion chromatography, at pH7.5, when 0.15mol/L sodium-chlor eluting, the respective peaks of purifying gained is collected with SDS-PAGE detection, the rhBMP2 that purity reaches the present invention of more than 95% can be obtained, each batch output is 359��378mg/L.
In addition, the encoding gene (Nucleotide is as shown in SEQIDNO:3) of the rhBMP2 of the code book invention that the optimization routine mode based on e. coli codon rate of utilization is obtained, carry out with reference to above method building, the rhBMP2 stoste expression and purification of the present invention obtain (purity SEC-HPLC%) >=95%, examine and determine through Nat'l Pharmaceutical & Biological Products Control Institute, meeting regulation,-terminal amino acid sequence results meets regulation.
The Bone formation experiment of the rhBMP2 of embodiment 2 the present invention
The rhBMP2 of the present invention and the BMP2 of No. 201410310953rd, Chinese patent application and No. 200910045832nd, Chinese patent application is adopted to carry out the differentiation calcification test of short bone. Specifically, get the bone marrow fluid of SD rat 1 (male), add in the DMEM nutrient solution containing 200ml/L foetal calf serum, 100U/ml penicillin and 100Lg/ml Streptomycin sulphate, with 37 DEG C, 50ml/L carbonic acid gas carry out cell cultures, nutrient solution is changed every 3 days, when merging 80% to marrow stromal cell, use 2.5g/L tryptic digestion, in 1:2 ratio Secondary Culture. Get the marrow stromal cell being passaged to for the 4th generation, with 104The amount in/hole is inoculated on 96 orifice plates, nutrient solution is abandoned after 24 hours, then it is divided into 4 groups at random and (on same 96 orifice plates, has whole each group, totally three piece of 96 orifice plate), every hole adds the DMEM nutrient solution (control group is not containing BMP2) of the different B MP2 containing 200ug/L, changed nutrient solution every 3 days, within 10,20 and 30 days, respectively take out one piece of 96 orifice plate in cultivation respectively and carry out Vonkossa dyeing, detect its gray-scale value and show differentiation calcification degree.
Result is as shown in table 1. Result shows, the BMP2 of the rhBMP2 of the present invention and No. 201410310953rd, Chinese patent application and No. 200910045832nd, Chinese patent application can urge bone differentiation calcification relative to control group, thus relative to control group, reduce the gray-scale value of Vonkossa dyeing more significantly. Wherein, the effect of the rhBMP2 of the present invention is best, has prospect in medicine most; Especially coordinating the optimization encoding gene of embodiment 1, the industrialization being applicable to very much genetically engineered is produced.
The Vonkossa dyeing gray-scale value of table 1 rat marrow stroma cell differentiation calcification
Claims (9)
1. human BMP-7, its aminoacid sequence is by such as nucleotide sequence coded shown in SEQIDNO:1.
2. polynucleotide, it encodes human BMP-7 according to claim 1.
3. polynucleotide according to claim 2, its nucleotide sequence is as shown in SEQIDNO:1.
4. carrier, it comprises the polynucleotide described in Claims 2 or 3.
5. cell, it comprises the polynucleotide described in Claims 2 or 3.
6. prepare the method for human BMP-7 according to claim 1, cultivate cell according to claim 5 when it is included in applicable protein expression, from culture, then isolate human BMP-7 according to claim 1.
7. pharmaceutical composition, it comprises human BMP-7 according to claim 1 and pharmaceutically acceptable carrier.
8. human BMP-7 according to claim 1 breaks up the application in the medicine of calcification at the short osteocyte of preparation.
9. application according to claim 8, wherein osteocyte is marrow stromal cell.
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| CN111269916B (en) * | 2020-03-17 | 2023-06-13 | 珠海深泓鑫生物科技有限公司 | Human bone morphogenetic protein 2 coding gene suitable for escherichia coli expression |
| CN114853867A (en) * | 2022-06-10 | 2022-08-05 | 上海交通大学医学院附属第九人民医院 | Preparation and application of polypeptide for promoting bone regeneration or bone formation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5508263A (en) * | 1988-10-11 | 1996-04-16 | Xoma Corporation | Heterodimeric osteogenic factor |
| CN104163865A (en) * | 2014-07-01 | 2014-11-26 | 杭州瑞固生物技术有限公司 | Optimized DNA sequences of recombinant human bone morphogenetic protein-2 and coded protein thereof |
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| CN104474591B (en) * | 2014-12-30 | 2017-05-03 | 筴文奎 | Bioactive degradable coral hydroxyapatite artificial bone |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5508263A (en) * | 1988-10-11 | 1996-04-16 | Xoma Corporation | Heterodimeric osteogenic factor |
| CN104163865A (en) * | 2014-07-01 | 2014-11-26 | 杭州瑞固生物技术有限公司 | Optimized DNA sequences of recombinant human bone morphogenetic protein-2 and coded protein thereof |
Non-Patent Citations (1)
| Title |
|---|
| 溶氧反馈-分批补料高密度培养人骨形成蛋白-2 工程菌;李毅，等;《生物工程学报》;20021130;第18卷(第6期);718-723 * |
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